Amino acid catabolism; Nucleic acid chemistry Andy Howard Introductory Biochemistry 29 April 2008.

Amino acid catabolism;

Nucleic acid chemistryAndy Howard

Introductory Biochemistry29 April 2008

29 April 2008Amino Acid metabolism p. 2 of 65

What we’ll cover

Amino acid catabolism Degradation products Interconversions Specifics

Urea cycle Reactions Cellular localization

Nucleic acid chemistry Pyrimidines: C, U, T Purines: A, G

Nucleosides Nucleotides Oligo- and

polynucleotides Duplex DNA Helicity RNA

structure types


What do we do with amino acids?

Obviously a lot of them serve as building-blocks for protein and peptide synthesis via ribosomal mechanisms

Also serve as metabolites, getting converted to other compounds or getting oxidized as fuel

Most amino acid degradations begin with transaminations to make glutamate; the resulting alpha-keto acids are further metabolized


Transaminations Generally two stages:

amino acid + -ketoglutarate -keto acid + glutamate

Glutamate + NAD+ + H2O -ketoglutarate + NADH + H+ + NH4

+

Net reaction isamino acid + NAD+ + H2O -keto acid + NADH + H+ + NH4

+


Glucogenic and ketogenic amino acids

Degradation of many amino acids lead to TCA cycle intermediates or pyruvate therefore these can be built back up to glucose; these are called glucogenic

Degradation of others leads to acetyl CoA and related compounds these cannot be built back up to glucose except

via the glyoxalate shuttle these are called ketogenic

Some amino acids are both!


Glucogenic amino acids

Amino acids that can be catabolized to produce building blocks that lead to glucose without help of glyoxalate pathway

Most produce succinate, succinyl CoA, fumarate, a-ketoglutarate, or oxaloacetate


Ketogenic amino acids

These do not produce TCA cycle intermediates, but rather produce acetyl CoA or its close relatives

Can be built back up into fats or ketone bodies


Serine-based metabolites

Serine is a building block for sphinganine and therefore for sphingolipids

Serine also leads to phosphatidylserine, which is important by itself and can be metabolized to phosphatidylethanolamine and phosphatidylcholine


Serine degradation

Two paths for degrading serine: PLP-dependent serine dehydratase

simply deaminates ser to pyruvate;this enzyme is like trp synthase

More common: SHMT transfers hydroxymethyl group to THF, leaving glycine; we’ve seen that one as a biosynthetic enzyme for making glycine

Serine dehydratasePDB 1P5J41 kDa monomerhuman


Glycine-based metabolites Glycine is a source for purines,

glyoxalate, creatine phosphate, and (with the help of succinyl CoA) porphobilinogen, whence we get porphyrins, and from those we get chlorophyll, heme, and cobalamin

porphobilinogen


Glycine cleavage system

Glycine + H2O + NAD+ + THF NADH + H+ + HCO3

- + NH4+ +

5-10-methyleneTHF Complex system: PLP,

lipoamide, FAD prosthetic groups Lipoamide swinging arm works

as in pyruvate dehydrogenase

T protein(aminomethyl-transferase) of glycine cleavage systemPDB 1V5V88 kDa dimerPyrococcus


asp, glu, ala degradation Standard transmination converts aspartate to

oxaloacetate with release of glutamate, which then can be deaminated to re-form -ketoglutarate: asp + -kg oxaloacetate + glu glu + NAD+ + H2O -kg + NADH + H+ + NH4+

Deamination converts glutamate to -ketoglutarate, as above

Standard transamination converts alanine to pyruvate according to the same logic as asp


All three of these are glucogenic! -ketoglutarate and oxaloacetate are

TCA cycle intermediates Pyruvate feeds the TCA cycle in ways

that can lead to glucose


Degradation of asn, gln

Asparagine and glutamine are deaminated to asp and glu

Thus they lead to oxaloacetate and -ketoglutarate, respectively

So they’re glucogenic The initial hydrolyses (deaminations)

are catalyzed by asparaginase and glutaminase

AsparaginasePDB 1O7J144 kDa tetramerErwinia chrysanthemi


Arginine degradation

Arginine is hydrolyzed to urea and ornithine as part of the urea cycle; enzyme is arginase

PLP-dependent enzyme converts ornithine to glu -semialdehyde

That’s oxidized to glutamate

ArginasePDB 2AEB212 kDa hexamerDimer shownHuman


Proline degradation

Proline oxidized back to 1-Pyrroline 5-carboxylate O2 is oxidizing agent different enzyme from forward

reaction Ring opened non-enzymatically

to form glutamate -semialdehyde; see arginine

Proline dehydrogenasePDB 2EKG72 kDa dimerThermus thermophilus


Histidine degradation

3 reactions from histidine toN-formiminoglutamate;first (HAL) makes urocanatefrom histidine

Tetrahydrofolate-dependent reaction produces glutamate and 5-formiminoTHF

5-formiminoTHF is enzymatically deaminated to 5,10-methyleneTHF, which can be used in purine synthesis, etc.

Histidine-ammonia lyasePDB 1GKM224 kDa tetramermonomer shownPseudomonas putida

urocanate


How are we doing so far?

We did ser and gly first because they’re so important

Then we’ve done a whole bunch that connect up to glutamate (or asp):asp, glu, ala, asn, gln, arg, pro, his

So we’re halfway through.


Threonine degradation

Several pathways (fig. 17.29) Major one: oxidize threonine to

2-amino-3-ketobutyrate 2-amino-3-keto-butyrate reacts

with HS-CoA to form acetyl CoA and glycine

So this one is ketogenic Other pathways are glucogenic

ThreoninedehydrogenasePDB 2DFV115 kDa trimerPyrococcus


Valine degradation (fig. 17.30, center)

Valine transaminated to-ketoisovalerate

Branched-chain -keto acid dehydrogenase (TTP, Lipoamide):-ketoisoavalerate + NAD+ + HS-CoA a-ketoisovaleryl CoA

Next reaction (acyl CoA dehydrogenase) 2-methyl-1-propenyl CoA + NADH + CO2

Product undergoes 4 reactions to propionyl CoA and thence to succinyl CoA: glucogenic

PDB 2VBF125 kDa dimerLacto-coccus


Isoleucine and leucine degradation

Same path but products are: Leucine’s products: acetyl CoA +

acetoacetate: ketogenic Isoleucine: Acetyl CoA + propionyl CoA:

ketogenic and glucogenic


Methionine degradation

A lot of methionine is turned intoS-adenosylmethionine: Methyl donor Leaves behind S-Adenosylhomocysteine

S-adenosylhomocysteine can be hydrolyzed to homocysteine and water

Homocysteine can condense with serine to form cystathionine, which can yield cysteine and -ketobutyrate… and we know how to turn -ketobutyrate into propionyl CoA. So met is glucogenic.


Cysteine degradation

Most common: oxidation to cysteinesulfinate, which transaminates to form -sulfinylpyruvate:cysteine + O2 cysteinesulfinate + H+

-sulfinylpyruvate undergoes nonenzymatic desulfuration to SO2 and pyruvate. So cysteine is glucogenic.

Cysteine dioxygenasePDB 2B5H22 kDa monomerrat

Cysteine-sulfinate


Phenylalanine Simple: phenylalanine

gets hydroxylated to form tyrosine:phenylalanine + O2 tyrosine

This is a tetrahydrobiopterin-dependent enzyme—a folate-like cofactor

Phenylalanine hydroxylasePDB 1J8U71 kDa dimermonomer shownhuman(residues 103-427)

Tetrahydro-biopterin


Phenylketonuria Usually associated with mutation in

phenylalanine hydroxylase: Accumulated Phe phenylpyruvate Afflicts 1/15000 newborns Built-up phenylpyruvate causes irreversible

mental retardation Type IV PKU related to deficiencies in

enzymes that restore tetrahydrobiopterin (see fig. 17.33, bottom)


Tyrosine degradation

Transaminated and mutated to homogentisate

Three more reactions convert that to fumarate + acetoacetate

So tyr (and phe) are both ketogenic and glucogenic

Homogentisate dioxygenasePDB 1EYB311 kDa hexamerMonomer shownHuman

homogentisate


Tryptophan degradation

Tryptophan: need to open 2 rings! 8 reactions lead to alanine and -

ketoadipate; first istrp + O2 -> N-formyl-kynurenine

Alanine gets transaminated to pyruvate

-ketoadipate goes through 6 more reactions to acetyl CoA + 2CO2

So it’s ketogenic and glucogenic

Indoleamine 2,3-dioxygenasePDB 2D0T89 kDa dimermonomer shownhuman


Lysine degradation (fig. 17.35)

Condense lysine with -ketoglutarate to form saccharopine

That’s deglutamated (?), oxidized, and transaminated to -ketoadipate

Six reactions degrade that to 2 acetyl CoA molecules plus 2 CO2

Purely ketogenic Some bacteria decarboxylate it to

cadaverine

SaccharopinedehydrogenasePDB 2AXQ103 kDa dimermonomer shownYeast


The urea cycle: overview

This is a significant pathway in the eukaryotic management of nitrogen-containing compounds

It was also one of the first biochemical pathways to be carefully characterized—by Krebs and coworkers!

Proceeds via ornithine & citrulline to urea and (in some organisms) uric acid

ornithine

urea


Making carbamoyl phosphate(fig. 17.37) Bicarbonate is phosphorylated to

form Ammonia condenses with that to

form carbamate and Pi

Second ATP-phosphorylation forms carbamoyl phosphate


Urea cycle itself In mitochondrion: carbamoyl phosphate

condenses with ornithine to form citrulline Citrulline condenses with urea to form

arginosuccinate Arginosuccinate is cleaved nonhydrolytically to

fumarate and arginine Arginine yields urea and citrulline Citrulline re-enters cycle


iClicker quiz: question 1 1. Glutamate + ammonia glutamine + H2O

is only slightly endergonic (Go’ = +14 kJ mol-1),yet it is coupled to ATP hydrolysis. Why?

(a) You can never run a reaction with a positive Go’

(b) [glutamate] ~ [glutamine] in the cell (c) If you heat the substrates, they disintegrate (d) ammonia is toxic in the absence of ATP


iClicker quiz #2

2. Which ribosomal amino acid’s biosynthesis is closely associated with the urea cycle? (a) alanine (b) serine (c) ornithine (d) arginine (e) none of the above.


Pyrimidines Single-ring nucleic acid bases 6-atom ring; always two nitrogens in the ring,

meta to one another Based on pyrimidine, although pyrimidine itself

is not a biologically important molecule Variations depend on oxygens and nitrogens

attached to ring carbons Tautomerization possible Note line of symmetry in pyrimidine structure

N

N

pyrimidine

1

2

3

4

5

6


Uracil and thymine Uracil is a simple dioxo

derivative of pyrimidine: 2,4-dioxopyrimidine

Thymine is 5-methyluracil Uracil is found in RNA;

Thymine is found in DNA We can draw other

tautomers where we move the protons to the oxygens

HN

OHN O

uracil

HN

O NH

O

thymine


Tautomers

Lactam and Lactim forms

Getting these right was essential to Watson & Crick’s development of the DNA double helical model

HN

OHN O

uracil - lactam

NH

ONO

uracil - lactimH

HN

O NH

O

thymine - lactam

HN

O N OH

thymine - lactim


Cytosine

This is 2-oxo,4-aminopyrimidine It’s the other pyrimidine base found in

DNA & RNA Spontaneous deamination (CU) Again, other tautomers can be drawn

N

OHN NH2

cytosine


Cytosine:amino and imino forms Again, this tautomerization needs to be

kept in mind

N

OHN NH

cytosine -imino form

N

OHN NH2

cytosine -amino form


Purines Derivatives of purine; again, the

root molecule isn’t biologically important

Six-membered ring looks a lot like pyrimidine

Numbering works somewhat differently: note that the glycosidic bonds will be to N9, whereas it’s to N1 in pyrimidines

HN

NN

N

purine

1

2

3

4

56 7

8

9


Adenine This is 6-aminopurine Found in RNA and DNA We’ve seen how important adenosine

and its derivatives are in metabolism Tautomerization happens here too

N

N

NH2

N

HN

adenine - amino form

HN

N

NH

N

HN

adenine - imino form


Guanine This is 2-amino-6-oxopurine Found in RNA, DNA Lactam, lactim forms

HN

NNH2N

HN

O

guanine - lactam

HN

NNH2N

N

OH

guanine - lactim


Nucleosides

As mentioned in ch. 8, these are glycosides of the nucleic acid bases

Sugar is always ribose or deoxyribose Connected nitrogen is:

N1 for pyrimidines (on 6-membered ring) N9 for purines (on 5-membered ring)

NR1R2

OH

HO

O

HO

N-glycoside of ribofuranose


Pyrimidine nucleosides Drawn here in amino and lactam forms

OH

OHHO

ON

ONH2N

cytidine

OH

OHHO

ON

ONH

O

uridine


Pyrimidine deoxynucleosides

OH

OHH

ON

ONH

O

2'-deoxyuridine

OH

OHH

ON

ONH

O

2'-deoxythymidineOH

OH

ON

ONH2N

deoxycytidine


A tricky nomenclature issue

Remember that thymidine and its phosphorylated derivatives ordinarily occur associated with deoxyribose, not ribose

Therefore many people leave off the deoxy- prefix in names of thymidine and its derivatives: it’s usually assumed.


Purine nucleosides

Drawn in amino and lactam forms

OH

HO

HO

O

N

N

NH2

N

N

adenosine

OH

HO

HO

O

N

N

O

HN

H2N N

guanosine


Purine deoxynucleosides

OH

HO

O

N

N

O

HN

H2N N

deoxyguanosine

OH

HO

O

N

N

NH2

N

N

deoxyadenosine


Chirality in nucleic acids Bases themselves are achiral Four asymmetric centers in

ribofuranose, counting the glycosidic bond.

Three in deoxyribofuranose Glycosidic bond is one of those 4 or 3. Same for nucleotides:

phosphates don’t add asymmetries


Mono-phosphorylated nucleosides

We have specialized names for the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates:

They are nucleotides Adenosine 5’-monophosphate =

AMP = adenylate GMP = guanylate CMP = cytidylate UMP = radiate

P

O

O-

O-O

HO

HO

O

N

N

NH2

N

N

adenylate


Deoxynucleotides Similar nomenclature

dAMP = deoxyadenylate

dGMP = deoxyguanylate

dCMP = deoxycytidylate

dTTP (= TTP) = deoxythymidylate = thymidylate

P

O

O-

O-O

HO

O

N

N

O

HN

H2N N

deoxyguanylate


Di and triphosphates Phosphoanhydride bonds link second and

perhaps third phosphates to the 5’-OH on the ribose moiety

OHHO

O

N

O

N

H2NP

O

O

O-O-

O

P

O

O-

O

P

O

OH

cytidine triphosphate

Mg2+


Oligomers and Polymers

Monomers are nucleotides or deoxynucleotides

Linkages are phosphodiester linkages between 3’ of one ribose and 5’ of the next ribose

It’s logical to start from the 5’ end for synthetic reasons


Typical DNA dinucleotide Various notations: this is pdApdCp Leave out the p’s if there’s a lot of them!

P

O

-O O-

O

O

NN

O

HN

NH2

N P

O

-O

O

O

O

ON

O N NH2

P

O--O

O


DNA structure

Many years of careful experimental work enabled fabrication of double-helical model of double-stranded DNA

Explained [A]=[T], [C]=[G] Specific H-bonds stabilize

double-helical structure: see fig. 19.12


What does double-stranded DNA really look like? Picture on previous slide emphasizes

only the H-bond interactions Fig.19.12 is better: shows the tilt of the

sugars Planes of the bases are almost

perpendicular to the helical axes on both sides of the double helix


Sizes (see fig. 19.14)

Diameter of the double helix: 2.37nm Length along one full turn:

10.4 base pairs = pitch = 3.40nm Distance between stacked base pairs =

rise = 0.33 nm Major groove is wider and shallower;

minor groove is narrower and deeper


What stabilizes this? Variety of stabilizing

interactions Stacking of base pairs Hydrogen bonding between

base pairs Hydrophobic effects (burying

bases, which are less polar) Charge-charge interactions:

phosphates with Mg2+ and cationic proteins

Courtesy dnareplication.info


How close to instability is it?

Pretty close. Heating DNA makes it melt: fig. 19.17 The more GC pairs, the harder it is to

melt Weaker stacking interactions in A-T One more H-bond per GC than per AT


iClicker quiz

3. What positions of a pair of aromatic rings leads to stabilizing interactions? (a) Parallel to one another (b) Perpendicular to one another (c) At a 45º angle to one another (d) Both (a) and (b) (e) All three: (a), (b), and ( c)


Final iClicker question!

4. Which has the highest molecular mass among the compounds listed? (a) cytidylate (b) thymidylate (c) adenylate (d) adenosine triphosphate (e) they’re all the same MW


Base composition for DNA

As noted, [A]=[T], [C]=[G] because of base pairing

[A]/[C] etc. not governed by base pairing Can vary considerably (table 19.2) E.coli : [A], [C] about equal Mycobacterium tuberculosis: [C] > 2*[A] Mammals: [C] < 0.74*[A]


Supercoiling Refers to levels of organization of DNA

beyond the immediate double-helix We describe circular DNA as relaxed if

the closed double helix could lie flat It’s underwound or overwound if the ends

are broken, twisted, and rejoined. Supercoils restore 10.4 bp/turn relation

upon rejoining: see fig. 19.19.


Supercoiling and flat DNA

Diagram courtesy SIU Carbondale


Ribonucleic acid We’re done with DNA for the moment. Let’s discuss RNA. RNA is generally, but not always, single-

stranded The regions where localized base-pairing

occurs (local double-stranded regions) often are of functional significance


RNA physics & chemistry RNA molecules vary widely in size, from a few

bases in length up to 10000s of bases There are several types of RNA found in cellsType %%turn- Size, Partly Role

RNA over by DS?

mRNA 3 25 50-104 no protein template

tRNA 15 21 55-90 yes aa activation

rRNA 80 50 102-104 no transl. catalysis &

scaffolding

sRNA 2 4 30-103 ? various

Amino acid catabolism; Nucleic acid chemistry Andy Howard Introductory Biochemistry 29 April 2008.

Documents

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